The major objective of the experiments described in this proposal is to elucidate the molecular mechanisms that control transcription of the ribosomal RNA genes in eukaryotes. The Xenopus rRNA genes are attractive to study not only because of their celluar importance, but also because they are well characterized in contrast, to most other eukaryotic genes. I have recently located the nucleotides which encode the 5' and 3' termini of the rRNA primary transcript on the RDNA sequence, and it is now possible to obtain faithful transcription of cloned rRNA genes. I propose to first simplify the determination of rDNA transcription from an electron microscopic to an electrophoretic assay. To this end I will construct a miniature rRNA gene (minigene) containing the rDNA initiation and termination regions but deleting most of the intervening 7500 base pairs. Next, I will locate the promoter and terminator sequences by making mutations in the minigene in vitro and assaying their effect on the transcription of the resulting plasmids. The proteins on rRNA genes will also be studied, first indrectly by their influence on the chromatin structure of the rDNA and later directly by their isolation and the characterization of their transcriptional effect. In particular, factors which regulate rRNA transcription will be sought and examined. The objective of the second project of this proposal is to determine whether the RNA that primes discontinuous DNA synthesis initiates at discrete sites on the SV40 genome or whether the "promoter" sequences for this RNA are not encoded in the DNA.